A preparation method of 2,4-difluorobenzylamine

Abstract

The present invention discloses a method for preparing 2,4-difluorobenzylamine, which comprises: contacting 2,4-difluorobenzonitrile with nitrile oxidoreductase, and reducing 2,4-difluorobenzonitrile to 2,4-difluorobenzylamine; wherein the nitrile oxidoreductase is the nitrile oxidoreductase with the NCBI accession number WP_004867005.1. The present invention also discloses an application of nitrile oxidoreductase in the preparation of dolutegravir or an intermediate thereof. The conversion rate of the preparation method of the present invention can reach 70%. This method has safe raw materials and a production process that is environmentally friendly, and is suitable for large-scale industrial production.

Description

Technical Field

[0001] The present invention relates to the field of biocatalysis, and particularly to a method for preparing a dolutegravir intermediate, namely 2,4-difluorobenzylamine. Background Art

[0002] Dolutegravir is a new anti-HIV drug under GlaxoSmithKline (GSK). On August 12, 2013, the US Food and Drug Administration (FDA) approved it for the treatment of HIV-1-infected adults who have been previously treated or are naïve, and children 12 years of age and older with a body weight of at least 40 kg. Due to its advantages of high efficiency and low toxicity, its market sales have increased rapidly in recent years, and the trend is unanimously optimistic.

[0003]

[0004] 2,4-Difluorobenzylamine is a key intermediate of dolutegravir, and its production method is very important. At present, the production method of 2,4-difluorobenzylamine mainly adopts the method of reducing 2,4-difluorobenzonitrile. For example, the method of reducing with hydrogen and Raney nickel is used in US5068371, but the yield is only 66%, and dangerous hydrogen needs to be used; another example is the method of hydrogenating 2,4-difluorobenzonitrile with a ruthenium precursor catalyst, a diphosphine ligand and 2-butanediol reported in Chemisitry A European Journal, 2013, 19(14), 4437-4440, but the yield is only 75%, and expensive metal catalysts need to be used, which causes heavy metal pollution to the environment. Summary of the Invention

[0005] In order to solve the technical problems in the prior art that the yield of preparing 2,4-difluorobenzylamine is low and the traditional chemical catalysis method causes environmental pollution, the present invention provides a method for preparing 2,4-difluorobenzylamine.

[0006] One of the technical solutions of the present invention for solving the above technical problems is: to provide a method for preparing 2,4-difluorobenzylamine, the preparation method comprising: contacting 2,4-difluorobenzonitrile with a nitrile oxidoreductase, and reducing 2,4-difluorobenzonitrile to 2,4-difluorobenzylamine; wherein the nitrile oxidoreductase is the nitrile oxidoreductase with the NCBI accession number WP_004867005.1.

[0007] Preferably, the amino acid sequence of the nitrile oxidoreductase has at least 90%, preferably at least 95%, 96%, 97%, 98%, more preferably at least 99% sequence identity with the nitrile oxidoreductase with the NCBI accession number WP_004867005.1, and has the function of reducing the nitrile-containing compound to 2,4-difluorobenzylamine.

[0008] In some preferred embodiments, the gene encoding the nitrile oxidoreductase comprises the nucleotide sequence shown in SEQ ID NO: 3.

[0009] In some preferred embodiments, the reaction conditions for the reduction include: pH is 7.2 - 7.5, and / or the temperature is 25 - 35 °C, preferably 30 °C.

[0010] In some preferred embodiments, the pH is adjusted with 20% aqueous sodium carbonate solution; and / or the reduction is carried out under oscillating conditions, preferably the oscillation is 200 rpm.

[0011] In some preferred embodiments, the reaction system for the reduction comprises a cosolvent, preferably isopropanol, and also comprises (1) a sufficient or excessive amount of reduced coenzyme, such as NADPH or NADH or FADH2 or FMNH2; or (2) a reduced coenzyme or an oxidized coenzyme and its regeneration system, and the regeneration system comprises a dehydrogenase and a hydrogen donor; preferably the oxidized coenzyme comprises NAD+, NADP+ or FAD or FMN, and the dehydrogenase and the hydrogen donor comprise one of alcohol dehydrogenase and isopropanol, glucose dehydrogenase and glucose, formate dehydrogenase and formate.

[0012] As is well known to those skilled in the art, the reduced coenzyme forms an oxidized coenzyme after one round of reaction, and the oxidized coenzyme is reduced to the reduced coenzyme in the presence of a dehydrogenase and a hydrogen donor, which can be recycled for the preparation method of the present invention.

[0013] In the present invention, the sufficient amount of reduced coenzyme means that the reduced coenzyme in the reaction system is theoretically sufficient for all 2,4-difluorobenzonitrile to be reduced to 2,4-difluorobenzylamine. Given a specific amount of 2,4-difluorobenzonitrile, those skilled in the art know how much reduced coenzyme should be added to make the catalytic reaction proceed fully. The excessive amount of reduced coenzyme means that the amount of reduced coenzyme in the reaction system exceeds the sufficient amount.

[0014] In some preferred embodiments, the regeneration system of the reduced coenzyme comprises NAD+ or NADP+, glucose dehydrogenase and glucose. Preferably, the glucose dehydrogenase is the forespore glucose 1-dehydrogenase of Bacillus subtilis subsp. More preferably, the glucose dehydrogenase comprises the amino acid sequence with NCBI accession number NP_388275.1. Even more preferably, the DNA encoding the glucose dehydrogenase comprises the nucleotide sequence with NCBI accession number NC_000964.3.

[0015] In some preferred embodiments, the nitrile oxidoreductase is prepared by the following steps:

[0016] (1) Inoculate the seed solution of nitrile reductase into a liquid medium containing antibiotics; preferably, the inoculation amount is 1 v / v%, the antibiotic is kanamycin with a final concentration of 50 μg / mL, and / or the liquid medium is LB or TB liquid medium;

[0017] (2) Cultivate until the OD 600 reaches 0.6 - 1.0, preferably 0.8, add IPTG for induction culture for 12 - 24 h, preferably 16 h, and then centrifuge to collect the bacterial cells; preferably, the cultivation temperature is 37 °C, the induction culture temperature is 25 °C, the final concentration of IPTG is 0.1 mM, and / or the centrifugation conditions are 4500 rpm for 20 min;

[0018] (3) Resuspend the bacterial cells in a buffer solution and homogenize and break them to obtain a crude enzyme solution; the buffer solution is preferably 0.05 M sodium phosphate buffer, pH 7.0;

[0019] Preferably, it further includes step (4): slowly add a 10% PEI flocculant with a total volume of 3 - 3.5% to the crude enzyme solution, stir for 5 - 30 min, preferably 10 min, and then centrifuge to take the supernatant; preferably, the centrifugation conditions are 4000 rpm for 20 min;

[0020] More preferably, it further includes step (0): inoculate an engineered bacterium containing the nitrile reductase gene into a liquid medium containing antibiotics for cultivation to obtain a seed solution; the antibiotic is kanamycin with a final concentration of 50 μg / mL, and / or the liquid medium is LB or TB liquid medium; preferably, the cultivation temperature is 37 °C.

[0021] In some preferred embodiments, the glucose dehydrogenase is prepared by the following steps:

[0022] (1) Inoculate the seed solution of glucose dehydrogenase into a liquid medium containing antibiotics; preferably, the inoculation amount is 1 v / v%, the antibiotic is kanamycin with a final concentration of 50 μg / mL, and / or the liquid medium is LB or TB liquid medium;

[0023] (2) Cultivate until the OD600 reaches 0.6 - 1.0, preferably 0.8, add IPTG for induction culture for 12 - 24 h, preferably 16 h, and then centrifuge to collect the bacterial cells; preferably, the cultivation temperature is 37 °C, the induction culture temperature is 25 °C, the final concentration of IPTG is 0.1 mM, and / or the centrifugation conditions are 4500 rpm for 20 min;

[0024] (3) Resuspend the bacterial cells in a buffer solution and homogenize and break them to obtain a crude enzyme solution; the buffer solution is preferably 0.05 M sodium phosphate buffer, pH 7.0;

[0025] Preferably, it further includes step (4) of slowly dripping 10% PEI flocculant with a total volume of 3 - 3.5% into the crude enzyme solution, stirring for 5 - 30 min, preferably 10 min, and then centrifuging to obtain the supernatant. Preferably, the centrifugation conditions are 4000 rpm for 20 min;

[0026] More preferably, it further includes step (0) of inoculating an engineered bacterium containing the glucose dehydrogenase gene into a liquid medium containing antibiotics for cultivation to obtain a seed solution; the antibiotic is kanamycin with a final concentration of 50 μg / mL, and / or the liquid medium is LB or TB liquid medium; preferably, the cultivation temperature is 37°C.

[0027] In some preferred embodiments, the concentration of 2,4-difluorobenzonitrile is 10 - 100 g / L, the mass ratio of nitrile reductase to 2,4-difluorobenzonitrile is 1:50 - 1:5, the molar ratio of glucose to 2,4-difluorobenzonitrile is 1:1 - 3:1, and the mass ratio of glucose dehydrogenase to glucose is 1:50 - 1:5; and / or, the mass ratio of reduced coenzyme or oxidized coenzyme such as NADPH or NADP+ to 2,4-difluorobenzonitrile is 1:20000 - 1:100.

[0028] To solve the above technical problems, the second technical solution of the present invention is: to provide an application of nitrile oxidoreductase in the preparation of dolutegravir or its intermediate; preferably, the intermediate is 2,4-difluorobenzylamine.

[0029] On the basis of conforming to common general knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0030] The reagents and raw materials used in the present invention are all commercially available.

[0031] The positive and progressive effects of the present invention are as follows:

[0032] Aiming at the problems of dangerous raw materials, high cost, and environmental pollution in the existing production process of 2,4-difluorobenzylamine, the present invention adopts a method of using nitrile reductase to catalytically reduce 2,4-difluorobenzonitrile to obtain 2,4-difluorobenzylamine, and the conversion rate can reach 70%. This method has safe raw materials, an environmentally friendly production process, and is suitable for large-scale industrial production. Specific Embodiments

[0033] The present invention will be further illustrated below by way of examples, but the present invention is not limited to the scope of the described examples. The experimental methods without specific conditions noted in the following examples are carried out according to conventional methods and conditions, or selected according to the product instructions.

[0034] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0035] The experimental methods in the present invention are all conventional methods unless otherwise specified. For the specific gene cloning operations, reference can be made to "Molecular Cloning: A Laboratory Manual" edited by J. Sambrook et al.

[0036] pET28a and the protein extraction reagent (bugbuster protein extraction reagent) were purchased from Novagen; DpnI enzyme was purchased from Invitrogen (Shanghai) Trading Co., Ltd.; NdeI enzyme and HindIII enzyme were purchased from Thermo Fisher; E. coli BL21(DE3) competent cells were purchased from Beijing Dingguo ChangSheng Biotechnology Co., Ltd.

[0037] Example 1 Preparation of nitrile reductase

[0038] Composition of LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L. Dissolve with deionized water and make up the volume. Sterilize at 121 °C for 20 min for later use.

[0039] Composition of TB liquid medium: peptone 10 g / L, yeast extract 18 g / L, glycerol 4 mL / L, KH 2 PO 4 2.31 g / L, K 2 HPO 4 ·2H 2 O 16.43 g / L. Dissolve with deionized water and make up the volume. Sterilize at 121 °C for 20 min for later use.

[0040] The amino acid and nucleotide sequence information of nitrile reductase is shown in Table 1 below. GenScript Biotech Corporation (No. 211 Pubin Road, Yanchuangyuan, Jiangbei New Area, Nanjing, Jiangsu Province) synthesized the genes of nitrile reductase (Enz.1 - Enz.3) (SEQ ID NO: 1 - 3) with the restriction enzyme sites NdeI and HindIII, and ligated them into the vector pET28a. The synthesized nitrile reductase genes were transformed into the host E. coli BL21(DE3) competent cells to obtain engineering strains containing the nitrile reductase genes.

[0041] Table 1 Information table of nitrile reductase

[0042]

[0043] After the engineered bacteria containing the nitrile reductase gene were separately activated by streaking on a petri dish, single colonies were picked and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured with shaking at 37 °C for 4 h. Transferred at an inoculation amount of 1 v / v% to 150 mL of fresh TB liquid medium containing 50 μg / mL kanamycin as well, and cultured with shaking at 37 °C until the OD 600 When it reached about 0.8, IPTG was added to a final concentration of 0.1 mM, and induced at 25 °C for 16 h. After the culture was completed, the culture solution was centrifuged at 4500 rpm for 20 min, the supernatant was discarded, the cells were collected, and stored in a -20 °C ultra-low temperature refrigerator for later use.

[0044] 10 g of the collected cells were taken and resuspended in 50 mL of 0.05 M sodium phosphate buffer at pH 7.0, and broken by high-pressure homogenization to obtain a crude nitrile reductase solution. 10% PEI flocculant with a total volume of 3 - 3.5% was slowly added dropwise to the crude enzyme solution, stirred for 10 min, centrifuged at 4000 rpm for 20 min, and the supernatant enzyme solution was taken to measure the nitrile reductase activity.

[0045] Method for measuring nitrile reductase activity:

[0046] 1 mL of reaction system at pH 7.4, 50 mM tris-Cl buffer, containing 15% DMSO, 0.1 mM NADPH, 0.1 mM substrate 2,4-difluorobenzonitrile, and an appropriate amount of enzyme solution. The consumption of NADPH was detected by spectrophotometry under the condition of a wavelength of 340 nm.

[0047] Definition of enzyme activity: Under the conditions of pH 7.4 and 30 °C, the amount of enzyme required to consume 1 μmoL NAD(P)H per minute is 1 enzyme activity unit (1 U).

[0048] Table 2 Liquid enzyme activity

[0049]

[0050] Among them, Enz.2 is the nitrile oxidoreductase disclosed in US7364882B1, which can catalyze the conversion of nitrile-containing compounds into corresponding amines (such as primary amines). However, the inventors found that Enz.2 cannot catalyze 2,4-difluorobenzonitrile to produce the dolutegravir intermediate 2,4-difluorobenzylamine.

[0051] Example 2 Preparation of glucose dehydrogenase

[0052] According to the glucose dehydrogenase (GDH) gene sequence derived from Bacillus subtilis 168 (NCBI accession number: NP_388275.1), GeneCreate BioLabs, Inc. (No. 211 Pubin Road, Yanchuang Park, Jiangbei New Area, Nanjing, Jiangsu Province) synthesized the GDH gene by total gene synthesis, with restriction enzyme sites NdeI and HindIII, and ligated it into the vector pET28a. The synthesized GDH gene was transformed into the host E. coli BL21(DE3) competent cells to obtain an engineered strain containing the GDH gene.

[0053] After the engineered bacteria containing the GDH gene were streaked and activated on a petri dish, single colonies were picked and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured with shaking at 37 °C for 4 h. Then, it was transferred to 150 mL of fresh TB liquid medium containing 50 μg / mL kanamycin at an inoculation amount of 1 v / v%, and cultured with shaking at 37 °C until the OD600 reached about 0.8. IPTG was added to a final concentration of 0.1 mM, and induced at 25 °C for 16 h. After the culture was completed, the culture solution was centrifuged at 4500 rpm for 20 min, the supernatant was discarded, the cells were collected, and stored in a -20 °C ultra-low temperature refrigerator for later use.

[0054] 10 g of the collected cells were resuspended in 50 mL of 0.05 M sodium phosphate buffer at pH 7.0, and broken by high-pressure homogenization to obtain a crude GDH enzyme solution. 10% PEI flocculant with a total volume of 3 - 3.5% was slowly added dropwise to the crude enzyme solution, stirred for 10 min, centrifuged at 4000 rpm for 20 min, and the supernatant enzyme solution was taken to measure the GDH enzyme activity. The protein concentration was measured using a Bradford kit (purchased from Shanghai Jierui Biotechnology Co., Ltd.).

[0055] GDH enzyme activity detection method: In a 1 mL reaction system, at 25 °C, first add 880 μL of 0.1 M disodium hydrogen phosphate - sodium dihydrogen phosphate buffer at pH 7.0 (containing 400 mM glucose), then add 100 μL of 25 mM NADP + solution, and finally add 20 μL of enzyme solution diluted 200 times. The OD value at 340 nm was measured using an ultraviolet spectrophotometer.

[0056] The enzyme activity is defined as: Under the conditions of pH 7.0 and 25 °C, the amount of enzyme required to generate 1 μmoL NAD(P)H per minute is 1 enzyme activity unit (1 U).

[0057] The measured enzyme activity was 1480 U / mL, and the protein concentration was 30 mg / mL.

[0058] Example 3 Preparation of 2,4-difluorobenzylamine by nitrile reductase catalyzing 2,4-difluorobenzonitrile

[0059]

[0060] Add 50 mL of pure water, 6.5 g of glucose, and 0.5 mg of NADP⁺ to the reaction system. Then add 20 mL of isopropanol and 5 g of 2,4-difluorobenzonitrile. Stir and dissolve in a 30 °C water bath. Fine-tune the pH to 7.2 - 7.5 with 20% sodium carbonate aqueous solution (mass / volume ratio). Finally, add 20 mL of the liquid nitrile reductase prepared in Example 1 (obtained by homogenizing 2.5 g of bacterial sludge with buffer) and 10 mL of GDH prepared in Example 2. Continuously react in a 30 °C water bath at pH 7.4 and 200 rpm. During the reaction process, detect the substrate conversion rate by HPLC. The results are shown in Table 3 below.

[0061]

[0062] As shown in the above table, the conversion rate of nitrile reductase Enz.03 in 20 hours is 70%. In this example, by adjusting the reaction system or reaction conditions, such as the reaction temperature being 20 - 40 °C and the amount of isopropanol being controlled at 5 - 50% of the total reaction volume, the preparation of 2,4-difluorobenzylamine can also be achieved. SEQUENCE LISTING <110> Yikele Biotech (Shanghai) Co., Ltd. <120> A method for preparing 2,4-difluorobenzylamine <130> P20016753C <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 843 <212> DNA <213> Vibrio cholerae <400> 1 atgtctaaat actctgacgc taaagaactg gcttctctga ccctgggtaa aaaaaccgaa 60 tacgctaacc agtacgaccc gtctctgctg cagccggttc cgcgttctct gaaccgtaac 120 gacctgcacc tgtctgctac cctgccgttc cagggttgcg acatctggac cctgtacgaa 180 ctgtcttggc tgaaccagaa aggtctgccg caggttgcta tcggtgaagt ttctatcccg 240 gctacctctg ctaacctgat cgaatctaaa tctttcaaac tgtacctgaa ctcttacaac 300 cagacccgtt tcgcttcttg ggacgaagtt cagacccgtc tggttcacga cctgtctgct 360 tgcgctggtg aaaccgttac cgttaacgtt aaatctctga acgaatacac cgctgaaccg 420 atcgttacca tgcagggtga atgcatcgac gaccaggaca tcgaaatcgc taactacgaa 480 ttcgacgacg ctctgctgca gggtgctgct cagggtgaag aagtttctga agttctgcac 540 tctcacctgc tgaaatctaa ctgcctgatc accaaccagc cggactgggg ttctgttgaa 600 atcgcttacc acggtgctaa aatgaaccgt gaagctctgc tgcgttacct ggtttctttc 660 cgtgaacaca acgaattcca cgaacagtgc gttgaacgta tcttcaccga catcatgcgt 720 tactgccagc cgcagtctct gaccgtttac gctcgttaca cccgtcgtgg tggtctggac 780 atcaacccgt tccgttcttc tcaccagtct gctccgaacc acaaccagcg tatggctcgt 840 cag 843 <210> 2 <211> 846 <212> DNA <213> Escherichia coli <400> 2 atgtcttctt acgctaacca ccaggctctg gctggtctga ccctgggtaa atctaccgac 60 taccgtgaca cctacgacgc ttctctgctg cagggtgttc cgcgttctct gaaccgtgac 120 ccgctgggtc tgaaagctga caacctgccg ttccacggta ccgacatctg gaccctgtac 180 gaactgtctt ggctgaacgc taaaggtctg ccgcaggttg ctgttggtca cgttgaactg 240 gactacacct ctgttaacct gatcgaatct aaatctttca aactgtacct gaactctttc 300 aaccagaccc gtttcaacaa ctgggacgaa gttcgtcaga ccctggaacg tgacctgtct 360 acctgcgctc agggtaaaat ctctgttgct ctgtaccgtc tggacgaact ggaaggtcag 420 ccgatcggtc acttcaacgg tacctgcatc gacgaccagg acatcaccat cgacaactac 480 gaattcacca ccgactacct ggaaaacgct acctgcggtg aaaaagttgt tgaagaaacc 540 ctggtttctc acctgctgaa atctaactgc ctgatcaccc accagccgga ctggggttct 600 ctgcagatcc agtaccgtgg tcgtcagatc gaccgtgaaa aactgctgcg ttacctggtt 660 tctttccgtc accacaacga attccacgaa cagtgcgttg aacgtatctt caacgacctg 720 ctgcgtttct gccagccgga aaaactgtct gtttacgctc gttacacccg tcgtggtggt 780 ctggacatca acccgtggcg ttctaactct gacttcgttc cgtctaccac ccgtctggtt 840 cgtcag 846 <210> 3 <211> 843 <212> DNA <213> Raoultella ornithinolytica <400> 3 atgtcttctt acgacaacca ccaggctctg gctggtctga ccctgggtaa atctaccgac 60 taccgtgaca cctacgacgc ttctctgctg cagggtgttc cgcgttctct gaaccgtgac 120 ccgctgggtc tgcacgctga caacctgccg ttccacggtg ctgacatctg gaccctgtac 180 gaactgtctt ggctgaacgc taaaggtctg ccgcagatcg ctgttggtca cgttgaactg 240 aacgacacca ccgttaacct ggttgaatct aaatctttca aactgtacct gaactctttc 300 aaccagaccc gtttcgctga ctggcaggaa gttgaagcta ccctgacccg tgacctgtct 360 gcttgcgctg aaggtgaagt tcaggtttct ctgtaccgtc tggacgaact ggaaggtcag 420 ccggttgctc acctgcacgg tgcttgcatc gacgaccagg acatcgaaat cgacaactac 480 cagttctctg ctgactacct gcaggacgct gcttctggta aagttgttga agaaaccctg 540 gtttctcacc tgctgaaatc taactgcctg atcacccacc agccggactg gggttctgtt 600 cagatccagt accgtggtgc taaaatcgac cgtgaaaaac tgctgcgtta cctggtttct 660 ttccgtcacc acaacgaatt ccacgaacag tgcgttgaac gtatcttcaa cgacatcctg 720 cgtttctgcc agccggaagc tctgtctgtt tacgctcgtt acacccgtcg tggtggtctg 780 gacatcaacc cgtggcgttc taacaccgac ttcgttccgg ctatcggtcg tctggctcgt 840 cag 843

Claims

1. A method for preparing 2,4-difluorobenzylamine, characterized in that, the preparation method comprises: contacting 2,4-difluorobenzonitrile with nitrile oxidoreductase, and reducing 2,4-difluorobenzonitrile to 2,4-difluorobenzylamine; wherein the nitrile oxidoreductase is the nitrile oxidoreductase with the NCBI accession number WP_004867005.1; the reaction system for the reduction comprises a cosolvent, and also comprises (1) a sufficient or excessive amount of reduced coenzyme, or (2) a reduced coenzyme or an oxidized coenzyme and its regeneration system, and the regeneration system comprises a dehydrogenase and a hydrogen donor; the reaction conditions for the reduction comprise: the pH is 7.2 - 7.5, and / or the temperature is 25 - 35°C.

2. The preparation method according to claim 1, characterized in that, the temperature is 30°C.

3. The preparation method as claimed in claim 1, characterized in that, the pH is adjusted with 20% aqueous sodium carbonate solution; and / or the reduction is carried out under oscillating conditions.

4. The preparation method according to claim 3, characterized in that, the oscillation is 200 rpm.

5. The preparation method as claimed in claim 1, characterized in that, the cosolvent is isopropanol, the reduced coenzyme is NADPH or NADH or FADH2 or FMNH2; the oxidized coenzyme comprises NAD+, NADP+ or FAD or FMN, and the dehydrogenase and the hydrogen donor comprise one of alcohol dehydrogenase and isopropanol, glucose dehydrogenase and glucose, formate dehydrogenase and formate.

6. The preparation method as claimed in claim 5, characterized in that, the regeneration system comprises glucose dehydrogenase and glucose.

7. The preparation method as claimed in claim 6, characterized in that, the glucose dehydrogenase is pre-spore glucose 1-dehydrogenase of Bacillussubtilis subsp.

8. The preparation method as claimed in claim 7, characterized in that, the glucose dehydrogenase comprises the amino acid sequence such as the NCBI accession number NP_388275.

1.

9. The preparation method as claimed in claim 7, characterized in that, the DNA encoding the glucose dehydrogenase comprises the nucleotide sequence such as the NCBI accession number NC_000964.

3.

10. The preparation method according to any one of claims 1 - 9, characterized in that, the nitrile oxidoreductase is prepared by the following steps: (1) Inoculating the seed liquid of the nitrile reductase into a liquid medium containing antibiotics; (2) Cultivate until OD 600 reaches 0.6 - 1.0, add IPTG and induce cultivation for 12 - 24 h, then centrifuge to collect the bacterial cells; (3) Resuspending the cell mass in a buffer solution, and homogenizing and crushing to obtain a crude enzyme solution.

11. The preparation method according to claim 10, characterized in that, in the step (1), the inoculation amount is 1 v / v%, the antibiotic is kanamycin with a final concentration of 50 μg / mL, and / or the liquid medium is LB or TB liquid medium; In (2) above, OD 600 is 0.8, the culture temperature is 37 °C, the induction culture time is 16 h, the temperature is 25 °C, the final concentration of IPTG is 0.1 mM, and / or the centrifugation conditions are 4500 rpm for 20 min; in the step (3), the buffer solution is 0.05 M sodium phosphate buffer solution with a pH of 7.

0.

12. The preparation method according to claim 10, characterized in that, The preparation method further includes step (4): slowly adding 10% PEI flocculant with a total volume of 3 - 3.5% to the crude enzyme solution, stirring for 5 - 30 min, and then centrifuging to obtain the supernatant.

13. The preparation method according to claim 12, wherein, in step (4), the stirring time is 10 min; the centrifugation conditions are 4000 rpm for 20 min.

14. The preparation method according to claim 10, wherein, the preparation method further includes step (0): inoculating an engineered bacterium containing a nitrile reductase gene into a liquid medium containing an antibiotic for cultivation to obtain a seed culture solution; the antibiotic is kanamycin with a final concentration of 50 μg / mL, and / or the liquid medium is LB or TB liquid medium.

15. The preparation method according to claim 14, wherein, the cultivation temperature in step (0) is 37°C.

16. The preparation method according to any one of claims 5 - 9, wherein, the glucose dehydrogenase is prepared by the following steps: (1) Inoculating a seed solution of glucose dehydrogenase into a liquid medium containing an antibiotic; (2) Cultivate until OD 600 reaches 0.6 - 1.0, add IPTG and induce cultivation for 12 - 24 h, then centrifuge to collect the thalli; (3) Resuspending the bacterial cells in a buffer solution and homogenizing and crushing to obtain a crude enzyme solution.

17. The preparation method according to claim 16, wherein, in (1), the inoculation amount is 1 v / v%, the antibiotic is kanamycin with a final concentration of 50 μg / mL, and / or the liquid medium is LB or TB liquid medium; In the above (2), OD 600 is 0.8; the culture temperature is 37 °C, the induction culture time is 16 h, the temperature is 25 °C, the final concentration of IPTG is 0.1 mM, and / or the centrifugation conditions are 4500 rpm for 20 min; in (3), the buffer solution is 0.05 M sodium phosphate buffer solution with a pH of 7.

0.

18. The preparation method according to claim 16, wherein, the preparation method further includes the step: (4) slowly adding 10% PEI flocculant with a total volume of 3 - 3.5% to the crude enzyme solution, stirring for 5 - 30 min, and then centrifuging to obtain the supernatant.

19. The preparation method according to claim 18, wherein, in (4), the stirring time is 10 min, and the centrifugation conditions are 4000 rpm for 20 min.

20. The preparation method according to claim 16, wherein, the preparation method further includes step (0): inoculating an engineered bacterium containing a glucose dehydrogenase gene into a liquid medium containing an antibiotic for cultivation to obtain a seed solution; the antibiotic is kanamycin with a final concentration of 50 μg / mL, and / or the liquid medium is LB or TB liquid medium.

21. The preparation method according to claim 20, wherein, in step (0), the cultivation temperature is 37°C.

22. The preparation method according to any one of claims 5 - 9, wherein, The concentration of the 2,4-difluorobenzonitrile is 10 to 100 g / L, the mass ratio of the nitrile reductase to the 2,4-difluorobenzonitrile is 1:50 to 1:5, the molar ratio of glucose to the 2,4-difluorobenzonitrile is 1:1 to 3:1, and the mass ratio of the glucose dehydrogenase to the glucose is 1:50 to 1:5; and / or, the mass ratio of the reduced coenzyme or oxidized coenzyme to the 2,4-difluorobenzonitrile is 1:20000 to 1:

100.

23. The preparation method according to claim 22, wherein, the reduced coenzyme or oxidized coenzyme is NADPH or NADP+.

24. Use of a nitrile oxidoreductase in the preparation of dolutegravir; the nitrile oxidoreductase has the NCBI accession number WP_004867005.1; the use comprises: contacting 2,4-difluorobenzonitrile with the nitrile oxidoreductase and reducing the 2,4-difluorobenzonitrile to 2,4-difluorobenzylamine; the reaction system for the reduction includes a cosolvent and also includes (1) a sufficient or excessive amount of reduced coenzyme, or (2) a reduced coenzyme or oxidized coenzyme and its regeneration system, and the regeneration system includes a dehydrogenase and a hydrogen donor; the reaction conditions for the reduction include: the pH is 7.2 to 7.5, and / or, the temperature is 25 to 35 °C.

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